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PubMed1994

Determination of thiamine and its phosphate esters in human erythrocytes by high-performance liquid chromatography with isocratic elution.

A high-performance liquid chromatographic method for the simultaneous determination of thiamine and its phosphate esters in human erythrocytes, using postcolumn derivatization, is presented. The sample preparation and the choice of the analytical column avoid the use of an elution gradient. The four thiamine compounds (thiamine and thiamine monophosphate, diphosphate and triphosphate) are eluted within less than 15 min with a detection limit of ca. 20 fmol. The reproducibility and accuracy of the assay are satisfactory. Normal physiological red blood cell concentrations of the four thiamine compounds are included.

Adult↗
PubMed1992

Orotidylate decarboxylase of yeast and man.

The mechanism for ODCase appears to involve the formation of a zwitterion of OMP and a ylid on decarboxylation. Thiamin pyrophosphate catalyzes various decarboxylation and transfer reactions involving ketone groups because the thiazolium ring with its positively charged N atom can, on the loss of a proton from the adjacent C-2, generate a ylid which adds to carbonyl groups to produce a substrate ylid. The unusual aspect, then, of the ODCase reaction is that the substrate itself becomes the ylid, presumably by gaining a proton from ODCase, which results in a positive charge on the N-1 atom of the pyrimidine ring. It is a zwitterion in the transition state which momentarily becomes a ylid on decarboxylation of OMP which then yields the product, UMP. There is no known cofactor for the ODCase reaction. It will be of interest to discover the groups on the enzyme that aid in formation of the zwitterion and the ylid. Further work on the crystal structure and on the production of altered enzymes (where specific amino acids suspected to be important for the reaction are changed) should reveal more details about this important and novel reaction.

Amino Acid Sequence↗
PubMed2002

A novel bifunctional molybdo-enzyme catalyzing both decarboxylation of indolepyruvate and oxidation of indoleacetaldehyde from a thermoacidophilic archaeon, Sulfolobus sp. strain 7.

An enzyme, which catalyzes both decarboxylation of indolepyruvate and subsequent oxidation of indoleacetaldehyde into indoleacetate, was purified from a thermoacidophilic archaeon, Sulfolobus sp. strain 7. The enzyme showed a M(r) of 280 kDa on gel filtration and was composed of three subunits (a, 89; b, 30; and c, 19 kDa), possibly in a stoichiometry of 2:2:2. Mo and Fe were detected. Thiamine pyrophosphate was absent. Biotin was suggested to bind to the b-subunit. The first step, the decarboxylation reaction, was specific for 2-oxoacids with an aromatic group, while in the second reaction, various aldehydes including glyceraldehyde, which is a glycolytic intermediate in the organism, were oxidized.

Aldehyde Oxidase↗
PubMed1988

Improved high-performance liquid chromatographic determination of thiamine and its phosphate esters in animal tissues.

An improved method for the determination of thiamine and its phosphate esters in animal tissues using reversed-phase high-performance liquid chromatography with precolumn derivatization is described. Thiamine and its phosphate esters were converted into fluorophores by alkaline cyanogen bromide, and the derivatives were applied to an ODS packed column. Then the effluent obtained by an acidic mobile phase was mixed with an alkaline methanol solution to increase the fluorescence intensity of the derivatives which was determined spectrofluorometrically. A complete, rapid and quantitative separation of thiamin and its phosphate esters was achieved and the use of the acidic buffer as a mobile phase improved the column stability. The fluorophores of thiochrome ester peaks on the chromatogram were sensitive to pretreatment with thiamine triphosphatase or acid phosphatase. The applicability of the method to the determination of the form of thiamin in various tissues of rat is demonstrated.

Animals↗
PubMed2001

Biosynthesis of menaquinone (vitamin K2) and ubiquinone (coenzyme Q): a perspective on enzymatic mechanisms.

The benzoquinone ubiquinone (coenzyme Q) and the naphthoquinones menaquinone (vitamin K2) and demethylmenaquinone are derived from the shikimate pathway, which has been described as a "metabolic tree with many branches." Menaquinone (MK) is considered a vitamin, but coenzyme (Q) is not; MK is an essential nutrient (it cannot be synthesized by mammals), whereas Q is not considered an essential nutrient since it can be synthesized from the amino acid tyrosine. The quinone nucleus of Q is derived directly from chorismate, whereas that of MK is derived from chorismate via isochorismate. The prenyl side chain of both quinones is derived from prenyl diphosphate, and the methyl groups are derived from S-adenosylmethionine. MK biosynthesis requires 2-ketoglutarate and the cofactors ATP, coenzyme A (CoASH), and thiamine pyrophosphate. In spite of the fact that both quinones originate from the shikimate pathway, there are important differences in their biosynthesis. In MK biosynthesis, the prenyl side chain is introduced in the next to last step, which is accompanied by loss of the carboxyl group, whereas in Q biosynthesis, the prenyl side chain is introduced at the second step, with retention of the carboxyl group. In MK biosynthesis, all the reactions of the pathway up to the prenylation (next to last step) are carried out by soluble enzymes, whereas all the enzymes involved in Q biosynthesis except the first are membrane bound. In MK biosynthesis the last step is a C-methylation; in Q biosynthesis, the last step is an O-methylation. In Q biosynthesis a second C-methylation and O-methylation take place in the middle part of the pathway. In spite of the fact that Q and MK biosynthesis diverges at chorismate, the C-methylations involved in both pathways are carried out by the same enzyme. Finally, Q biosynthesis under aerobic conditions requires molecular oxygen; anaerobic biosynthesis of Q and MK incorporates oxygen atoms derived from water. The current status of the pathways with particular emphasis on the reaction mechanisms, is discussed in this review.

Escherichia coli↗
PubMed1989

Nervous system damage and nutritional factors in chronic alcoholics.

Dietary habits in 45 chronic alcoholics were assessed by the dietary history method, and plasma albumin and serum transferrin. These variables as well as clinical findings did not indicate general malnutrition, but a moderate deficiency in folate intake was found. There was no correlation between the nutritional variables and central and peripheral nervous system function investigated by neuroradiological, neuropsychological, and neurophysiological methods. Transketolase and thiamine-pyrophosphate effects were measured in a subgroup of alcoholics and compared to a control group. No differences were found and there was no correlation to central nervous system impairment. These findings do not support the hypothesis that nervous system damage found in chronic alcoholics is of nutritional etiology.

Adult↗
PubMed1989

Optimized determination of thiochrome derivatives of thiamine and thiamine phosphates in whole blood by reversed-phase liquid chromatography with precolumn derivatization.

A liquid chromatographic method involving precolumn derivatization for determining thiamine and its phosphate esters in human blood has been optimized. Blood sample stored at - 20 degrees C were haemolysed and deproteinized by perchloric acid. The supernatants of the samples were oxidized by addition of potassium ferricyanide-sodium hydroxide, and phosphoric acid was added to obtain a neutral pH in order to extend the column life. The samples were stable after derivatization for at least 24 h, if protected from light and kept at room temperature. Gradient separation with 140 mmol phosphate buffer (pH 7.0), and methanol, tert-butylammonium hydroxide and dimethylformamide as modifiers, on a 3-microns Chromsphere octadecylsilica column gave an analysis time of 15 min. The method was found to be very suitable for the determination of thiamine components in whole blood. The minimal detectable amount is 0.5 nmol/l and the method is linear to at least 1000 nmol/l. The recovery (98 +/- 3%) and precision are very good.

Chromatography, Liquid↗
PubMed1993

The oxygenase reaction of acetolactate synthase.

In addition to the physiological reactions catalyzed by acetolactate synthase, it supports an oxygen-consuming side reaction. Although the synthase and oxygenase activities are activated to somewhat different extents by various metals (Mn2+, Mg2+, Ca2+, Co2+, Zn2+, Ni2+, Cd2+, Cu2+, Ba2+, Al3+), the modest degree of these differences (at most 6-fold) and the high degree of promiscuity of the enzyme with respect to its metal requirement suggest that the metal is not intimately involved in the chemistry of either reaction. Saturation of the oxygenase reaction occurs at pyruvate concentrations below the limit of sensitivity for the oxygen electrode (< 10 microM), at higher concentrations pyruvate inhibits the rate of oxygen consumption. At a noninhibitory concentration of pyruvate (1 mM), inhibition of the reaction is also observed with alpha-ketobutyrate. Inhibition of the oxygenase reaction by high concentrations of pyruvate or alpha-ketobutyrate is presumably due to competition between these substrates and molecular oxygen for a common carbanionic reaction intermediate, the conjugate base of (hydroxyethyl)thiamin pyrophosphate. Inhibition of the reaction indicates that the lactylthiamin pyrophosphate intermediate can decarboxylate prior to binding of the second pyruvate or alpha-ketobutyrate. At high concentrations of pyruvate or alpha-ketobutyrate, only incomplete inhibition of the oxygenase reaction is achieved (65-89% or 89-93% maximal inhibition, respectively). This incomplete inhibition of the oxygenase reaction by alpha-keto acids indicates that the reaction is not Theorell-Chance with respect to addition of the second alpha-keto acid and that oxygen has more than one route of access to the carbanionic reaction intermediate.

Acetolactate Synthase↗
PubMed1994

Characterization of an ancestral type of pyruvate ferredoxin oxidoreductase from the hyperthermophilic bacterium, Thermotoga maritima.

The hyperthermophilic bacterium, Thermotoga maritima, is a strict anaerobe that grows up to 90 degrees C by carbohydrate fermentation. We report here on its pyruvate ferredoxin oxidoreductase (POR), the enzyme that catalyzes the oxidation of pyruvate to acetyl-CoA, the terminal oxidation step in the conversion of glucose to acetate. T. maritima POR was purified to electrophoretic homogeneity under strictly anaerobic conditions. It has a molecular weight of 113,000 and comprises four dissimilar subunits with M(r) values of approximately 43,000, 34,000, 23,000, and 13,000. It contains thiamine pyrophosphate (TPP) and at least two ferredoxin-type [4Fe-4S] clusters per molecule, as determined by iron analysis and EPR spectroscopy. CoASH was absolutely required for pyruvate oxidation activity, while the addition of TPP was stimulatory. The apparent Km values at 80 degrees C for pyruvate, CoASH, and TPP were 14.5, 0.34, and 0.043 mM, respectively, and the corresponding apparent Vm values ranged from 154 to 170 mumol of pyruvate oxidized/min/mg (units/mg). The apparent Km and Vm values for T. maritima ferredoxin, the proposed physiological electron carrier for POR, were 26 microM and 280 units/mg, respectively. POR did not use 2-oxoglutarate, phenyl pyruvate, or indolyl pyruvate as substrates. The enzyme was extremely thermostable: the temperature optimum for pyruvate oxidation was above 90 degrees C, and the time for a 50% loss of activity (t50%) at 80 degrees C (under anaerobic conditions) was 15 h. The enzyme was also very sensitive to inactivation by oxygen, with a t50% in air at 25 degrees C of 70 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1983

Heterogeneity of binding sites for the pyruvate dehydrogenase component on the dihydrolipoyl transacetylase core of bovine kidney pyruvate dehydrogenase complex.

We have characterized the dissociation equilibrium constant (Kd) and the rate constants of association and dissociation for the binding of the pyruvate dehydrogenase component (PDH) to the dihydrolipoyl transacetylase component of kidney pyruvate dehydrogenase complex. We have found about 7 high-affinity sites (Kd = 1.5 X 10(-11) M) and about 13 low-affinity sites (Kd = 2.5 X 10(-8) M) with negative cooperativity for binding of PDH at the weaker sites. The high-affinity sites show a much higher rate constant for association of PDH with the core than do the low-affinity sites. Catalytic turnover strengthens binding of PDH at high-affinity sites when the complex is preincubated in the presence of thiamin pyrophosphate (TPP). In the absence of TPP, appreciably tighter binding occurs at the low-affinity sites (Kd = 5.9 X 10(-10) M). TPP weakens PDH binding at these sites with a half-maximal effect at about 5 microM TPP. In addition to TPP, increased ionic strength or 1-3 mM Mg2+ (with an enhanced effect of Mg2+ at higher ionic strengths) weakens PDH binding at low-affinity sites with a corresponding increase in the rate constant for dissociation. Further studies will be required to determine whether site heterogeneity contributes to dynamic processes in the function and regulation of the pyruvate dehydrogenase complex.

Acetyltransferases↗
PubMed1984

Bromopyruvate as an active-site-directed inhibitor of the pyruvate dehydrogenase multienzyme complex from Escherichia coli.

Bromopyruvate behaves as an active-site-directed inhibitor of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase complex of Escherichia coli. It requires the cofactor thiamin pyrophosphate (TPP) and acts initially as an inhibitor competitive with pyruvate (Ki ca. 90 microM) but then proceeds to react irreversibly with the enzyme, probably with the thiol group of a cysteine residue. E1 catalyzes the decomposition of bromopyruvate, the enzyme becoming inactivated once every 40-60 turnovers. Bromopyruvate also inactivates the intact pyruvate dehydrogenase complex in a TPP-dependent process, but the inhibition is more rapid and is mechanistically different. Under these conditions, bromopyruvate is decarboxylated, and the lipoic acid residues in the lipoate acetyltransferase (E2) component become reductively bromoacetylated. Further bromopyruvate then reacts with the new thiol groups thus generated in the lipoic acid residues, inactivating the complex. If reaction with the lipoic acid residues is prevented by prior treatment of the complex with N-ethylmaleimide in the presence of pyruvate, the mode of inhibition reverts to irreversible reaction with the E1 component. In both types of inhibition of E1, reaction of 1 mol of bromopyruvate/mol of E1 chain is required for complete inactivation, and all the evidence is consistent with reaction taking place at or near the pyruvate binding site.

Affinity Labels↗
PubMed1984

Inhibition of pyruvate dehydrogenase multienzyme complex from Escherichia coli with a radiolabeled bifunctional arsenoxide: evidence for an essential histidine residue at the active site of lipoamide dehydrogenase.

Incubation of pyruvate dehydrogenase multienzyme complex (PD complex) from Escherichia coli with thiamin pyrophosphate, pyruvate, coenzyme A, Mg2+, and the radiolabeled bifunctional arsenoxide p-[(bromoacetyl)-amino]phenyl arsenoxide (BrCH214CONHPhAsO) led to the irreversible loss of lipoamide dehydrogenase (E3) activity. The mode of inactivation occurred by initial "anchoring" of the reagent via its -AsO group to reduced lipoyl residues on lipoate acetyltransferase (E2) (generated by substrates) followed by the delivery of the BrCH214CO- moiety into the active site of E3 where an irreversible alkylation ensued [Stevenson, K. J., Hale, G., & Perham, R. N. (1978) Biochemistry 17, 2189]. To account for nonspecific alkylations, not mediated by this delivery process, control experiments were conducted in which the radiolabeled bifunctional reagent was incubated with PD complex in the absence of substrates. E3 subunits were isolated from inhibited and control PD complexes by chromatography on hydroxylapatite in the presence of 8 M urea. Acid hydrolysis of the alkylated E3 and control E3 samples produced radiolabeled carboxymethylated amino acids that were identified and quantitated by high-voltage electrophoresis and amino acid/radiochemical analysis. The inhibited sample contained N3-(carboxymethyl)histidine and a small amount of S-(carboxymethyl)cysteine. These residues were not present in significant amounts in the controls. The loss of 81% of E3 activity correlated with the alkylation of about 0.7 residue of histidine and 0.1 residue of cysteine per mol of E3.

Alkylation↗
PubMed1984

Elementary steps in the reaction mechanism of the alpha-ketoglutarate dehydrogenase multienzyme complex from Escherichia coli: kinetics of succinylation and desuccinylation.

The kinetics of the succinylation and the desuccinylation of the alpha-ketoglutarate dehydrogenase complex from Escherichia coli have been studied at 4 degrees C in 2 mM thiamin pyrophosphate, 2 mM MgCl2, and 20 mM potassium phosphate (pH 7.0) by steady-state and quenched-flow techniques. The initial steady-state velocity for the reaction of the complex is inhibited by high concentrations of alpha-ketoglutarate. The data are consistent either with cooperative interactions between two catalytic sites or with the existence of an alpha-ketoglutarate regulatory site. The time course of the succinylation by alpha-ketoglutarate of the unmodified complex or the complex in which a fraction of the alpha-ketoglutarate decarboxylase subunits (E1) has been inhibited with N-ethylmaleimide reveals a complex kinetic process. A mechanism consistent with the kinetic data is proposed in which some E1 subunits succinylate one lipoic acid per E1 and other E1 subunits succinylate two lipoic acids per E1. Furthermore, each succinylation reaction occurs via a two-step process with rate constants of 49 and 89 s-1 at saturating concentrations of alpha-ketoglutarate for the first and second steps, respectively. At long times, 13-16 mol of succinate binds per mol of unmodified complex. The stoichiometry of binding obtained with N-ethylmaleimide-treated complex is initially lower but approaches the same values as for the unmodified complex over the course of minutes. Coenzyme A removes the succinyl groups on the unmodified enzyme with a rate constant greater than or equal to 200 s-1. The results obtained suggest a limited accessibility between sites on the complex.

Escherichia coli↗
PubMed1985

Purification and properties of Salmonella typhimurium acetolactate synthase isozyme II from Escherichia coli HB101/pDU9.

A facile purification has been devised for recombinantly produced Salmonella typhimurium acetolactate synthase isozyme II. Purification of the enzyme was made possible by determining the complex set of factors that lead to loss of enzymic activity with this rather labile enzyme. When complexed with thiamin pyrophosphate, FAD, and magnesium, acetolactate synthase is subject to oxygen-dependent inactivation, a property not shared by the enzyme-FAD complex. When divorced from all of its tightly bound cofactors, losses of the enzymic activity are encountered at low ionic strength, especially at low protein concentrations. If purified and stored as the enzyme-FAD complex, acetolactate synthase is quite stable. The enzyme is composed of two types of subunits, a result that was not anticipated from previous studies of ilvG (the gene that codes for the large subunit of acetolactate synthase). These subunits were determined to be in equal molar ratio in the purified enzyme from the distribution of radioactivity between the two subunits after carboxymethylation with iodo[14C]acetate and their respective amino acid compositions. Besides the expected ilvG gene product (59.3 kDa), purified acetolactate synthase contained a smaller subunit (9.7 kDa; designated here as the ilvM gene product). On the basis of sequence homology of the small subunit with that coded for by the corresponding Escherichia coli gene sequence [Lawther, R. P., Calhoun, D. H., Adams, C. W., Hauser, C. A., Gray, J., & Hatfield, G. W. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 922-925], it is encoded by the region between ilvG and ilvE, beginning at base-pair (bp) 1914 (relative to the point of transcription initiation).(ABSTRACT TRUNCATED AT 250 WORDS)

Acetolactate Synthase↗
PubMed1988

Kinetics and mechanism of benzoylformate decarboxylase using 13C and solvent deuterium isotope effects on benzoylformate and benzoylformate analogues.

Benzoylformate decarboxylase (benzoylformate carboxy-lyase, BFD; EC 4.1.1.7) from Pseudomonas putida is a thiamine pyrophosphate (TPP) dependent enzyme which converts benzoylformate to benzaldehyde and carbon dioxide. The kinetics and mechanism of the benzoylformate decarboxylase reaction were studied by solvent deuterium and 13C kinetic isotope effects with benzoylformate and a series of substituted benzoylformates (pCH3O, pCH3, pCl, and mF). The reaction was found to have two partially rate-determining steps: initial tetrahedral adduct formation (D2O sensitive) and decarboxylation (13C sensitive). Solvent deuterium and 13C isotope effects indicate that electron-withdrawing substituents (pCl and mF) reduce the rate dependence upon decarboxylation such that decreased 13(V/K) effects are observed. Conversely, electron-donating substituents increase the rate dependence upon decarboxylation such that a larger 13(V/K) is seen while the D2O effects on V and V/K are not dramatically different from those for benzoylformate. All of the data are consistent with substituent stabilization or destabilization of the carbanionic intermediate (or carbanion-like transition state) formed during decarboxylation. Additional information regarding the mechanism of the enzymic reaction was obtained from pH studies on the reaction of benzoylformate and the binding of competitive inhibitors. These studies suggest that two enzymic bases are required to be in the correct protonation state (one protonated and one unprotonated) for optimal binding of substrate (or inhibitors).

Benzoates↗
PubMed1990

Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli.

The genes that encode the five known enzymes of the mandelate pathway of Pseudomonas putida (ATCC 12633), mandelate racemase (mdlA), (S)-mandelate dehydrogenase (mdlB), benzoylformate decarboxylase (mdlC), NAD(+)-dependent benzaldehyde dehydrogenase (mdlD), and NADP(+)-dependent benzaldehyde dehydrogenase (mdlE), have been cloned. The genes for (S)-mandelate dehydrogenase and benzoylformate decarboxylase have been sequenced; these genes and that for mandelate racemase [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540] are organized in an operon (mdlCBA). Mandelate racemase has regions of sequence similarity to muconate lactonizing enzymes I and II from P. putida. (S)-Mandelate dehydrogenase is predicted to be 393 amino acids in length and to have a molecular weight of 43,352; it has regions of sequence similarity to glycolate oxidase from spinach and ferricytochrome b2 lactate dehydrogenase from yeast. Benzoylformate decarboxylase is predicted to be 499 amino acids in length and to have a molecular weight of 53,621; it has regions of sequence similarity to enzymes that decarboxylate pyruvate with thiamin pyrophosphate as cofactor. These observations support the hypothesis that the mandelate pathway evolved by recruitment of enzymes from preexisting metabolic pathways. The gene for benzoylformate decarboxylase has been expressed in Escherichia coli with the trc promoter, and homogeneous enzyme has been isolated from induced cells.

Acetolactate Synthase↗
PubMed1981

Inhibition of pyruvate dehydrogenase multienzyme complex from Escherichia coli with a bifunctional arsenoxide: selective inactivation of lipoamide dehydrogenase.

The bifunctional reagent p-[(bromoacetyl)-amino]phenyl arsenoxide (BrCH2CONHPhAsO) in the presence of excess reduced nicotinamide adenine dinucleotide has been shown to cause the irreversible active site directed inactivation of the lipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase multienzyme (PD) complex from Escherichia coli. The ability of the lipoate acetyltransferase (E2) component to bind coenzyme A was decreased by about 50% in this system. In the presence of thiamine pyrophosphate, pyruvate, coenzyme A, and Mg2+, E3 inactivation by BrCH2CONHPhAsO was selective (coenzyme A binding was unaffected) and stoichiometrically related to PD complex inactivation, indicating that a complement of E3 is necessary for full complex activity. The activity of the pyruvate dehydrogenase (E1) component was unaltered by BrCH2-CONHPhAsO in both systems. On inhibition of the PD complex with BrCH2CONHPhAsO, the reagent mediated interchain cross-linking between E2 and about half of the E3 subunits. A marked change occurred in the quaternary structure of the PD complex, with some E1 and E3 subunits being dissociated from the E2 core. The mechanism outlined by Stevenson et al. [Stevenson, K. J., Hale, G., & Perham, R. N. (1978) Biochemistry 17, 2189] for the inhibition of the PD complex by BrCH2CONHPhAsO must be revised on the basis of these findings. E3 is only partially modified by delivery of the bromoacetyl moiety of the bifunctional reagent (covalently attached to lipoyl residues of E2 through dithioarsinite bonds) into the active site of bound E3. The inhibition of E3, dissociated from the PD complex during cross-linking, likely occurs via direct interaction of the free enzyme with BrCH2CONHPhAsO by initial dithioarsinite modification of the reduced active-site disulfide followed by alkylation of a nearby residue.

Arsenicals↗